Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.     

Uptake of75Se in cataractous rat lens proteins

The purpose of the present study was to measure the pattern of uptake of⁷⁵Se into proteins in normal rat lenses and into the proteins of lenses with selenite-induced cataract. Ten-day-old suckling rats received a single injection of⁷⁵Se with or without a cataractous dose of cold carrier sodium selenite. Four days after injection, the proteins from excised lenses were counted for⁷⁵Se radioactivity and subjected to gel permeation chromatography, amino acid analyses, and mass spectrometry. All three soluble crystallin lens proteins took up⁷⁵Se in both normal and cataractous lenses. However, cataractous lenses did not take up⁷⁵Se into a soluble protein in which major quantities of⁷⁵Se were taken up in normal rats. Futhermore,⁷⁵Se in the gamma-crystallins was associated with an unusual acidic amino acid. It was concluded that selenium metabolism by lens proteins may be unusual compared to other soft tissues.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.     

Immunochemical comparison of the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Expression of the major intrinsic protein (MIP) of eye-lens fibre cell membranes was compared in normal (DBA), cataractous (CAT, LOP, NCT) and chimaeric (CBA-LOP) mice at different stages of development using immunofluorescence microscopy and immunoblotting techniques. MIP of apparent molecular mass 26 kDa was detected in extracts of adult DBA, LOP and CBA-LOP lenses, but only low molecular mass (less than 26 kDa) immunoreactive proteins were detected in similar extracts from adult CAT and NCT lenses. The corresponding MIP distribution patterns confirmed the highly organised fibre-cell histology in embryonic DBA and adult CBA-LOP lenses and also highlighted the severe fibre-cell degeneration in the LOP lens. In contrast, however, no immunoreactive MIP was detected in situ in embryonic CAT and NCT lenses. These results suggest that a structural alteration of MIP occurs during embryonic lens development in the cataractous CAT (dominant) and NCT (recessive) mutant mice.     

Role of the non-enzymatic glycosylation of protein in the formation of human cataracts

We have measured the free epsilon amino groups in soluble and insoluble proteins of clear human lenses and diabetic and non-diabetic senile cataractous lenses. The free epsilon amino groups content of soluble and insoluble proteins was significantly lower in diabetic cataracts than in clear lenses and non diabetic senile cataracts. Our results seem to demonstrate that non-enzymatic glycosylation of lens protein could play a role in the pathogenesis of cataract in diabetes.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Temporal relationship between lens protein oxidation and cataract development in streptozotocin-induced diabetic rats

We compared the progression of lens opacification with the time course of oxidation of lens proteins under conditions of streptozotocin-induced experimental diabetes in rats. By the end of the 17th week, approx. 50% of the diabetic animals developed mature cataracts. During the following month, 95% of the eyes in the diabetic group became cataractous. In the course of lens opacification we observed a time-dependent increase in the content of protein carbonyls and decrease in the concentration of protein sulfhydryls in the lenses of diabetic animals. Significantly higher protein carbonyl (p<0.01) and lower protein sulfhydryl (p<0.001) content was found in lenses with the advanced stage of cataract when compared with the diabetic lenses still transparent. We showed that the values of protein carbonyls exceeding 1.2 nmol/mg protein and of sulfhydryls falling below 60 nmol/mg protein corresponded to an approximately 50% incidence of mature cataract development. At the end of the 34th week, when all lenses of diabetic rats became cataractous, the corresponding values of protein carbonyls and sulfhydryls were 2.5 nmol/mg protein and 27 nmol/mg protein, respectively. The main finding of this study is the disclosure of quantitative relationship between the degree of protein oxidation and the rate of advanced cataract development in the widely used model of streptozotocin-induced experimental diabetes in rats.     

Lens glutathione, lens protein glycation and electrophoretic patterns of lens proteins in STZ induced diabetic rats

As diabetes is a very complex disease, with the pathological symptoms varying with age, diabetic type and means of control, it still warrants manyin vivo andin vitro studies. During hyperglycaemia, increases in the sorbitol pathway, nonenzymatic glycosylation of lens proteins and damage to antioxidant systems have been reported to cause opacification of the lens leading to cataract formation. In this study, intracapsular extracts of lenses from STZ induced diabetic female rats were examined. Total protein, glutathione and nonenzymatic glycosylation were determined by the Lowry, Ellman reagent and thiobarbituric acid methods respectively. Laemmli protein electrophoresis was also carried out on the lens homogenates. After a period of as short as 5 weeks, a decrease in lens glutathione, and an increase in nonenzymatic glycosylation of lens proteins were found. The electrophoresis showed an increase in proteins of high molecular weight.     

Localization of neutral and acidic glycosphingolipids in rat lens

Rat lens was found to contain several neutral and acidic glycosphingolipidsin lens epithelia, cortex and nucleus, and showed developmentalchanges in their content and localization. TLC-immunostainingof gangliosides revealed the enrichment of some ganglio-seriesgangliosides (GM3, GM1, GD3 and GD1b) in lens epithelia andthe presence of GM3 and GD3 in the lens nucleus. Immunohistochemicalstudies confirmed the distribution of GM3 and GM1 in anteriorlens epithelial cells and the cortex, with expression decreasingtoward the lens nucleus. Immunoreaction to GD3 was more intensein the lens nucleus than in epithelial cells. In contrast, theexpression of neolacto-series glycosphingolipids was restrictedto the lens nucleus. In order to investigate the pathologicalchanges of glycosphingolipids in cataract, galactose-inducedcataractous lenses were examined. However, no significant changeswere observed in the content and composition of glycosphingolipids.In addition, Lewis^x epitopes found in human cataractous lenseswere not detected in the cataractous lenses of galactosaemicrats and hereditary cataractous Emory mice. cataract gangliosides glcosphingolipids Lewis^x rat lens     

用蛋白质印迹转移技术分析正常及硒性白内障大鼠晶状体及房水中蛋白质的性质

本文用蛋白质印迹转移技术分析了正常及硒性白内障大鼠晶状体及房水中蛋白质的性质。结果表明,晶状体中的脲溶性蛋白质可被抗α及抗γ晶体蛋白血清识别,提示α及γ晶体蛋白均为脲溶性蛋白质的主要成份。患白内障时房水中的蛋白质含量明显增加,且主要被抗γ血清识别,而被抗α血清识别的成份很少,表明在大鼠硒性白内障形成过程中,有较多低分子量蛋白质漏出到房水中,且其主要成份为γ晶体蛋白。此外,我们还发现正常及硒性白内障大鼠晶状体膜蛋白质与抗α及抗γ血清起反应的程度及分布有所不同,提示晶状体细胞膜与晶体蛋白之间存在着相互作用。     

Peroxide-metabolizing systems of the crystalline lens

The ability of transparent and cataractous human, rabbit and mice lenses to metabolize hydrogen peroxide in the surrounding medium was evaluated. Using a chemiluminescence method in a system of luminol-horseradish peroxidase and a photometric technique, the temperature-dependent kinetics of H₂O₂ decomposition by lenses were measured. The ability of opaque human lenses to catalyze the decomposition of 10^?4 M H₂O₂ was significantly decreased. However, this was reserved by the addition of GSH to the incubation medium. Incubation of the mice lenses with the initial concentration H₂O₂ 10^?4 M led to partial depletion of GSH in normal and cataractous lenses. Human cataractous lenses showed decreased activities of glutathione reductase, glutathione peroxidase (catalyzing reduction of organic hydroperoxides including hydroperoxides of lipids), superoxide dismutase, but no signs of depletion in activities of catalase or glutathione peroxidase (utilizing H₂O₂). The findings indicated an impairment in peroxide metabolism of the mature cataractous lenses compared to normal lenses to be resulted from a deficiency of GSH. An oxidative stress induced by accumulation of lipid peroxidation products in the lens membranes during cataract progression could be considered as a primary cause of GSH deficiency and disturbance of the redox balance in the lens.     

Defensive Role of Quercetin Against Imbalances of Calcium,Sodium, and Potassium in Galactosemic Cataract

Galactosemic cataracts are characterized by electrolyte disturbances resulting in osmotic imbalance and loss of transparency. We have studied the defensive role of quercetin, a bioflavonoid, against the alterations of calcium (Ca²⁺), sodium (Na⁺), and potassium (K⁺) concentrations in galactose-induced cataract in a rodent model. The experimental study was conducted on weanling male Wistar rats with an average body weight of 34 ± 0.9 g. Different groups received normal stock AIN 93 diet (group A, n = 8), AIN 93 diet with 30% galactose (group B, n = 8), and AIN 93 diet with 30% galactose + quercetin at 400 mg/100 g diet (group C, n = 8). Aldose reductase activity and protein content and concentrations of Ca²⁺, Na+, and K⁺ were determined in normal and cataractous lenses. Treatment with quercetin resulted in a significant decrease in Na⁺ and Ca²⁺ and aldose reductase levels and an increase in K⁺ and protein levels in galactosemic cataractous lenses. These results imply that inclusion of quercetin contributes to lens transparency through the maintenance of characteristic osmotic ion equilibrium and protein levels of the lens.     

Elevated tropomyosin expression is associated with epithelial–mesenchymal transition of lens epithelial cells

Injury to lens epithelial cells (LECs) leads to epithelial–mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α‐smooth muscle actin (α‐SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up‐regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non‐cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α‐SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm‐based inhibitors for postponing PCO and cataractogenesis.     

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography.     

Glucosylation of human lens protein and cataractogenesis.

Examination of glucosylation of lens protein was conducted utilizing tritiated BH₄^?. The overall results indicate that approximately 0.20 moles of tritium were incorporated per mole of protein. Similar results were obtained with normal and senile cataractous lenses with varying degrees of opacity. Furthermore no difference in the ³H incorporation was observed between soluble and insoluble protein fractions derived from these lenses. Investigation of selected polypeptides isolated from the senile cataracts gave comparable results. Protein isolated from diabetic lenses had only slightly higher levels of tritium incorporation, giving an average value of 0.27 moles per mole of protein. Analyses of the tritiated products indicate that approximately 50% of the incorporation is probably due to reduction of other types of compounds. These results suggest that glucosylation does not appear to be a primary factor in cataract formation.     

Distinct energy requirement for nuclear import and export of importin beta in living cells

Carbohydrates with reactive aldehyde and ketone groups can undergo Maillard reactions with proteins to form advanced glycation end products. Oxalate monoalkylamide was identified as one of the advanced glycation end products formed from the Maillard reaction of ascorbate with proteins. In these experiments, we have analyzed human lens proteins immunochemically for the presence of oxalate monoalkylamide. Oxalate monoalkylamide was absent in most of the very young lenses but was present in old and cataractous lenses. The highest levels were found in senile brunescent lenses. Incubation experiments using bovine lens proteins revealed that oxalate monoalkylamide could form from the ascorbate degradation products, 2,3-diketogulonate and L-threose. These data provide the first evidence for oxalate monoalkylamide in vivo and suggest that ascorbate degradation and its binding to proteins are enhanced during lens aging and cataract formation.     

Are the proteinase inhibitory activities in lenticular tissues real?

The possibility of proteinase inhibitory activities in lenses measured with synthetic substrates being spurious, due to the effective competition of lens proteins as substrates for the target enzymes, was investigated. Goat, sheep and human cataractous lens proteins were found to be poor substrates for trypsin, elastase and papain compared to casein or bovine serum albumin. Further, the inhibition of elastase catalyzed hydrolysis of succinyl trialanyl p-nitroanilide by casein (500 μg, 53%) and albumin (500 μg, 49%) and of trypsin-catalyzed hydrolysis of benzoyl argininep-nitroanilide by albumin (1 mg, 24%) were significant only at high protein concentrations. These data indicated that the relatively high antielastase and antitryptic activities observed in human cataractous lenses were real. On the other hand, coincident lens protein hydrolysis elevating the true antitryptic and antielastase activities in goat and sheep lenses (that have low activities) could not be ruled out The lesser papain inhibitory activities observed in lenses when albumin was used as substrate compared to activities with benzoyl arginine p-nitroanilide as substrate, appeared to be partly due to lens protein hydrolysis masking the actual inhibition in the former method. Preincubation of goat, sheep and human lens extracts with trypsin for 1 h resulted in complete loss of antitryptic and antielastase activity except in the case of human lens antielastase activity which underwent 50% loss. Papain inhibitory activity was fully stable. Similar papain treatment caused loss of 80–100% of antielastase activity and 45–55% loss of antitryptic activity.     

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n?=?52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n?=?11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.     

Comparison of post-translational modifications of alpha A-crystallin from normal and hereditary cataract rats

Summary. In order to investigate the relationship between lens opacities and the various modifications of lens proteins, we analyzed and compared the properties of lens proteins of 85-day old normal Wistar rats and the hereditary cataract model, ICR/f rats. The present study identified many differences between normal and mutant lens proteins. In the ICR/f mutant rats, the relative amounts of gamma-crystallin decreased and high molecular weight (HMW) protein increased. Racemization and isomerization of Asp-151 of alpha A-crystallin was observed in the mutant ICR/f rats, and Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. These modifications were not found in the age-matched normal rats. These tendencies are consistent with aged and cataractous human lenses.     

UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products: IMPLICATIONS FOR HUMAN LENS AGING AND CATARACT FORMATION*

Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm², 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans.